The present invention relates to the field of chemical synthesis and, more particularly, to novel coupling agents that can be beneficially utilized in the syntheses of various substances and particularly in peptide synthesis.
All known life forms and life processes are based on proteins which are essential to all biological functions. Consequently, all the illnesses and disorders associated with life involve proteins. Among many other functions, proteins play a key role in signaling pathways of immunological and/or neurological processes and thus they are major players in many congenital, chronic and infectious diseases and disorders.
As such, proteins exhibit highly potent therapeutic efficacy and therefore attract a great pharmaceutically-focused attention, especially in the post-genomic and proteomic era, where important advances in molecular biology and gene technology are bringing significant changes in biomedical sciences. The elucidation of the human genome, followed by advancements in functional genomics and proteomics, revolutionized the understanding of the detailed molecular mechanisms, underlying a broad spectrum of peptide and protein related diseases and disorders. As a result, novel therapeutic targets have been identified and novel mechanism-based therapeutic paradigms have been suggested, whereby peptides play a key role in all of these transitions.
Consequently there has been a notable increase in researches, both academic and industrial, which are aimed at developing novel peptide-based drugs. From a pharmaceutical market point of view, peptides are presently considered as active pharmaceutical ingredients (API) that can be used in a variety of therapeutic applications and particularly as, for example, antibiotics, hormones, immunomodulators, anti-angiogenesis agents, therapeutic agents for treating CNS and other neurological disorders, analgesics, anti-obesity drugs, and as therapeutic agents for treating immune disorders such as allergy, asthma, hemophilia, anemia and autoimmune diseases [Loffet, A. 2002, J Peptide Sci. 8, 1].
However, although peptides have enormous therapeutic potential, their widespread use has been limited by several restrictive technical factors. Today, manufacturing companies face the unprecedented challenge of producing hundred kilograms to tons quantities of complex peptides. Such a massive production typically uses expensive and complex modern technologies, rendering peptide manufacture difficult and cost-inefficient as compared with other pharmaceuticals. Large-scale manufacturing and purification of peptides in a bioactive form can therefore be a limiting step in the commercialization of peptide-based drugs.
A key step in the peptide production process is the formation of the peptide bond (an amide bond formed between the carboxylic acid group of one amino acid and the amine group of another amino acid). In peptide syntheses, formation of a peptide bond typically requires the activation of the carboxylic acid, which usually involves the use of a peptide coupling agent [for a comprehensive review on peptide coupling agents see, F. Albericio, S. A. Kates, Solid-Phase Synthesis: A Practical Guide, S. A. Kates, F. Albericio Eds; Marcel Dekker, New York, N.Y., 2000, pp. 273-328 and F. Albericio, R. Chinchilla, D. J. Dodsworth, C. Najera, 2001, Org Prep. Proc. Int., 33, 202].
Although the synthesis of medium-large peptides for basic research is a well established procedure, the combination of the 20 naturally occurring amino acids and a growing number of unnatural amino acids makes each peptide synthesis unique at the industrial level, oftentimes requiring closer attention to each amino acid coupling. Some of the rules for coupling agents validated in the research scale can be applied at industrial level, but the results are still hardly predictable.
Thus use of peptide coupling agents in the industry has started as early as 1955 with the introduction of dicyclohexylcarbodiimide (DCC) [Sheehan, J. C. and Hess, G. P., 1955, J. Am. Chem. Soc., 77, 1067], which at that time was already known and well studied as a reagent for formation of amide bond [Khorana, H. G., 1952, J. Chem. Soc., 2081]. The chemical structures of DCC, as well as of 1-ethyl-3-(3′dimethylaminopropyl)carbodiimide (EDC) and N,N′-diisopropylcarbodiimide (DIC), other coupling agents of the carboiimide family, are presented in FIG. 1.
Carbodiimides are indeed the most available and low-cost coupling agents amongst the presently known reagents. The primary reactive species, O-acylisourea, is one of the most reactive species for peptide coupling. Shortcomings associated with the use of carbodiimides as coupling agents therefore mostly stem from the high and relatively incontrollable reactivity thereof and include, for example, racemization, side reactions and low yields due to the formation of the poorly active N-acyl urea. Furthermore, while low dielectric constant solvents such as CHCl3 or CH2Cl2 are optimal for carbodiimides, the use of these solvents precludes the use of automatic solid-phase synthesis machines, and thus, carboiimides coupling agents can be used, at most, in manual or semi-automatic modes. Carbodiimides are also incompatible with Fmoc/t-Bu solid-phase chemistry, because the urea derivative formed in such syntheses is typically not soluble in common solvents. Such urea derivatives are also difficult to remove in solution chemistry.
At the beginning of the 70's, 1-hydroxybenzotriazole (HOBt) [König, W., Geiger, R., 1970, Chem. Ber. 103, 788 and 2034] was proposed as an additive to DCC. The addition of HOBt was aimed at reducing the racemization associated with DCC coupling. The relative success of this additive signaled the beginning of a period during which other benzotriazole derivatives such as 1-hydroxy-56-chlorobenzotriazole (Cl-HOBt) [Ueki, M. and Yanagihira, T., Peptides 1998, Proceedings of the 25th European Peptide Symposium, S. Bajusz, F. Hudecz Eds, Akademiai Kiado, Budapest, p. 252] and 1-hydroxy-7-azabenzotriazole (HOAt) [Carpino, L. A., 1993, J. Am. Chem. Soc., 115, 4397] have been developed and successfully used (see, FIG. 2). During the past years the addition of benzotriazole derivatives as additives to DCC and other carbodiimides became almost mandatory to preserve the peptide bond formation by carbodiimide activation of low yields and undesired side reactions and loss of chirality.
In the last decade, the use of phosphonium and aminium/uronium salts, referred to herein in short as “onium salts”, of hydroxybenzotriazole derivatives as peptide coupling agents, has been introduced. Although these reagents have been rapidly adapted for research purposes, only a few of them have been found compatible with current industrial requirements and synthetic strategies and were adopted by the industry.
The species that reacts with onium salts is the carboxylate of the amino/organic acid. Therefore, performing the coupling reaction in the presence of at least one equivalent of a base is essential while using these reagents. The mechanism by which these “onium” salts acts as coupling agents is schematically illustrated in FIG. 3.
The presently most reactive aminium salt coupling agent is 2-(7-aza-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HATU) [Carpino, L. A., 1993, J. Am. Chem. Soc., 115, 4397]. The chemical structures of HATU and analogs thereof are presented in Scheme 1 below.
SCHEME 1 XYZHATUHNPF6HBTUHCHPF6TBTUHCHBF4HCTUClCHPF6TCTUClCHBF4
However, the use of the aminium salts as peptide coupling agents was found to be limited in various aspects. First, during the activation of hindered carboxylic components, such as those involved in cyclization reactions of hindered amino acids, the aminium salts can react with a more available amino component, leading to a guanidine derivative, a process that terminates the peptide chain. Furthermore, it has been discovered that aminium salts can contain traces of dimethylamine, which can also react with the carboxylic component to give the corresponding dimethylamide. Finally, HATU as well as its less reactive analogues (see, Scheme 1 above) are prohibitively expensive, rendering their industrial use impractical.
The most prevalent phosphonium salts of benzotriazoles that are presently used as peptide coupling agents are benzotriazol-1-yl-N-oxy-tris(pyrrolidino)phosphonium hexafluorophosphate (PyBOP) [Coste, J. et al., 1975, Tetrahedron Lett., 31, 205] and the later uncovered 7-azabenzotriazol-1-yl-N-oxy-tris(pyrrolidino)phosphonium hexafluorophosphate (PyAOP) [Albericio, F. et al., 1997, Tetrahedron Lett., 38, 4853], which is derived from HOAt and is presently considered as the most reactive phosphonium salt (see, FIG. 4). PyBOP and PyAOP, are specifically useful as coupling agent in cyclization steps or for the activation of hindered amino acids, where the use of aminium salts can lead to the formation of guanidine derivatives. Since it was found that PyBOP and PyAOP do not terminate the peptide chain, these coupling agents can be used in excess and be added during the coupling step, while circumventing the risk of chain termination.
Thus, the presently known peptide coupling agents are typically limited by their low desired reactivity, side products formed thereby and/or high cost.
Due to the ongoing developments of peptide-based drugs and the limitations associated with the presently known peptide coupling agents, there is a widely recognized need, and it would be highly advantageous to have novel, efficient peptide coupling agent devoid of the above limitations.
It is noteworthy that coupling agents that are useful in peptide synthesis can also be used in other organic syntheses that require activation of a carboxylic moiety. Such syntheses can be used to produce organic compounds of biological interest such as, for example, peptoids, oligocarbamates, oligoamides, β-lactams, esters, polyenamides, benzodiazepines, diketopiperazines, and hydantoins.